Method of producing citric acid by fermentation



United States PatentO IVIETHOD OF PRODUCING CITRIC ACID BY FERMENTATIONLeonard B. Schweiger, Elkhart, Ind., assignor to Miles Laboratories,Inc., Elkhart, Ind., a corporation of Indiana N Drawing. Application'July 8, 1957 Serial No. 670,390

Claims. (Cl. 195-36) This invention relates to the production of citricacid from carbohydrate-containing materials by submerged vegetativefermentation methods, and more particularly relates to a method ofinhibiting certain contaminating effects in the fermentation mediumwhich interfere with citric acid production. This application is acontinuation-in-part of my application Serial No. 358,950, filed June 1,1953, and now abandoned.

Although the sugar-containing media conventionally used in citric acidproduction by submerged fermentation methods are known to have a highresistance to contamination by most micro-organisms, the same media areconversely recognized as being susceptible to contamination from strainsof penicillia and certain yeasts. This immunity to the first form ofcontamination and susceptibility to the latter is primarily attributableto the low pH of the substrate media which is optimumly from about 2.2to 2.8.

The major effect of the yeast contamination is in reduced fermentationefficiency resulting from competitive utilization of the sugar in themedia by both the yeast and the acid producing mold such as Aspergillusniger. However, the presence of penicillia in early of late stages offermentation has a more absolute effect in that it causes a completecessation of acid production. Though it is commonly referred to asstaling of the medium, this inability to yield the prescribed amount ofcitric 2,916,420 Patented Dec. 8, 1959 Aspergillus niger have achievedthe widest acceptance in submerged fermentation methods because of theirhigh citric acid producing properties.

To preliminarily establish the inhibitory effects of penicillia, and inorder to determine the time required for a definite number of organismsto produce staling of a fermentation medium, decationized corn sugarmedia were inoculated with various concentrations of penicillia sporesat the time when the initial A. niger inoculation was made and thefollowing staling effects were noted:

Table I Penicillia Spores/Liter Contamination Days +=Suflicieutpenicillia growth to alter acid production.

Since a conventional fermentative conversion of a medium normallycontinues over a period of time of about eight to ten days, it can beseen from Table I that all concentrations of penicillia spores down toas little as ten per liter of the medium caused a staling effect withinthe course of the fermentation period. Penicillia contamination in allexamples was established by microscopic examination, fermentation odorand lack of acid production. As vcan beseen, the time required for thepenicillia to effect a staling or reduction of acid production desolublein the fermentation medium such as cupric sulacid is not due to changein the medium but" appears to be brought about through a parasiticrelationship between the contaminant, penicillia, and the fermentativeorganisms.

Accordingly, it is an object of this invention to elimi natefermentation losses due to penicillia contamination.

It is a further object of this invention to provide a method ofpreventing the development of' penicillia incidentally present insubmerged fermentation media undergoing vegetative conversion to citricacid.- I

A further object of this invention is to eliminate the inhibitory effectof penicillia without altering the growth and physiological activity offermentative organisms.

These and other related objects are achieved by .my invention wherein Ihave eliminated the undesirable effect of penicillia by inclusion ofcopper in the fermentation medium at a point in the fermentation processafter the growth of the Aspergillus niger has reached a stage that isoptimum for acid yields. It has been found that, under ordinarycircumstances, this optimum growth of Aspergillus niger is establishedbetween about 40 and 50 hours after commencement of the fermentation,Le, 40 to 50 hours after inoculation.

In order to illustrate the concepts of my. invention in greater detail,reference will be made to experiments and tests on media containingAspergillus niger as the fermen-tative organism, said media being soconstituted as to conform with those optimum conditions set forth inU.S. Patents Nos. 2,492,667 and 2,492,673, and being subjected tofermentation procedures therein described. As is commonly known, certainstrains and mutants of fate, cupric chloride, cuprous chloride, cupricnitrate, cuprous cyanide, cuprous iodide, cupriccarbonate, cupricacetate, and the like.

Table II Penicillia Cu Observations 1 Batch A nidity,

No. g.

Spores] Days p.p.m. Days Gontam- Days liter ination 1 +=Suificientpenicillia growth to alter acid production. =No eviconcentrations ofboth penicillia spores and copper ions and amount of medium which hadundergone fermentative conversion for 2 /3 days, varying amounts ofpenicillia spores were added as shown in batches 2, 3, 4, and 5, and theeifect on acid production and staling time were noted. It can be seenthat these four experiments show a somewhat longer time to staling thanthose experiments tabulated in Table I for the reason that these fourwere not contaminated during the initial 2 /3 days of fermentation.Under the same circumstances but with amounts of copper varying betweenS and 75 p.p.m. being added simultaneously with the artificialcontamination of the medium by penicillia spores, acid production inbatches 6 through 9 was greatly increased with no observablecontamination of the medium being evident. The same improved acidproduction and absence of observable contamination in the final mediumwere noted in batches 10 through 14 wherein artificial contamination bypenicillia spores was eifected at the onset of fermentation, and whereincopper was introduced after 2% and 3% days.

It is evident from these tabulations that in every fermentation to whichionic copper was added, penicillia did not develop to the point ofcausing a decrease in acid production. Moreover, those fermentationswhere contamination was induced, and to which no ionic cooper was added,stopped producing acid completely. In this latter series offermentations it is to be incidentally noted that ionic copper did notmaterially affect the acid yields.

The following data further illustrate the present process:

Fermentations were commenced in three commercialscale citric acidfermentors and portions of the media used therein were withdrawn andplaced, respectively, in laboratory fennentors. The media wereconstituted and the fermentations carried out in accordance with theprocedure described in US. Patents 2,492,667 and 2,492.673. Copper wasadded to the commercial-scale fermentors after the optimum growth of thefermenting organism, A. niger, had been established and no penicilliacontamination resulted. In the corresponding laboratory fermen- T tors,no copper was added; all the latter fermentors 'became staled, and noacid was produced. These data are set out in Table III below:

Although the exact mechanism of this selective inhibitory effect of theionic copper is not known, the data set forth in Tables II and III,above, demonstrate that, once the proper cellular morphology of theAspergillus niger for citric acid production has been established,amounts of copper between about 5 and about 200 parts per million partsof the fermenting medium do not affect the acid producing rate of thefermenting organism, but effectively inhibit the penicillia. ever, thatif copper is added to a fermenting medium at a time prior to about 40 to50 hours after inoculation, the copper has an adverse effect on acidyields.

In view of these fermentation results in which ionic copper successfullyeliminated losses due to penicillia contamination, ionic copper wasadded to two fully fermented batches which were known to be contaminatedand which had lost their acid-producing capability.

I have also found, how- It can be seen from comparison of the finalamount of acid obtained after addition of the ionic copper, as comparedwith the percent acid in the medium condemned as being incapable offurther fermentation, that both of these fermentation media were broughtback into production. Inasmuch as the normal acid concentration in afully fermented medium approximates 10 percent, full utilization ofstate fermentation media can now be achieved according to the methods ofmy invention. It will be understood that the data in Table IV areillustrative, and that copper additions throughout the range of 5200pants per million parts of the media are effective to revivefermentations made stale by penicillia. contamination.

Having thus described my invention, I claim:

1. In the production of citric acid by submerged fermentation withAspergillus niger of a carbohydrate-containing medium, the method ofinhibiting penicillia growth and development in said medium whichcomprises incorporating into said fermentation medium, after developmentof the proper cellular morphology of the Aspergillus niger forproduction of citric acid, ionic copper in amounts between about 5 andabout 200 parts per million parts of fermentation medium.

2. In a process for the production of citric acid by the submergedfermentation with Aspergillus niger of a carbohydrate-containing medium,the method of inhibiting penicillia growth and development in saidmedium which comprises incorporating into said fermentation medium,after from 40 to 50 hours of fermentation, ionic copper in amountsbetween about 5 and about 200 parts per million parts of fermentationmedium.

3. A process in accordance with claim 2, wherein said copper is suppliedby a copper salt soluble in said fennentation medium.

4. A process in accordance with claim 3, wherein said coppersalt isselected from the group consisting of cupric sulfate, cupric nitrate,cuprous chloride, cupric chloride, cuprous cyanide, cuprous iodide,cupric carbonate, and cupric acetate.

5-. A method of reviving citric acid production with Aspergillus nigerin a submerged fermentation medium having further acid productioninhibited by penicillia. development therein, which method comprisesincorporating in said medium ionic copper, the proportion of said copperbeing between about 5 and about 200 parts thereof per million parts ofsaid medium.

References Cited in the file of this patent UNITED STATES PATENTS2,353,771 Szucs July 18, 1944 2,364,701 Eisenman et al Dec. 12, 1944OTHER REFERENCES The Botanical Review by J. W. Foster, No. 4, April

1. IN THE PRODUCTION OF CITRIC ACID BY SUBMERGED FERMENTATION WITHASPERGILLUS NIGER OF A CARBOHYDRATE CONTAINING MEDIUM, THE METHOD OFINHIBITING PENICILLIA GROWTH AND DEVELOPMENT IN SAID MEDIUM WHICHCOMPRISES INCORPORATING INTO SAID FERMENTATION MEDIUM, AFTER DEVELOPMENTOF THE PROPER CELLULAR MORPHOLOGY OF THE ASPERGILLUS NIGER FORPRODUCTION OF CITRIC ACID, IONIC COPPER IN AMOUNTS BETWEEN ABOUT 5 ANDABOUT 200 PARTS PER MILLION PARTS OF FERMENTATION MEDIUM.